Antigen Binding Proteins that Bind CXCR3

ABSTRACT

There is disclosed compositions and methods relating to or derived from anti-CXCR3 antibodies. More specifically, there is disclosed fully human antibodies that bind CXCR3, CXCR3-binding fragments and derivatives of such antibodies, and CXCR3-binding polypeptides comprising such fragments. Further still, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having CXCR3 related disorders or conditions, including various inflammatory disorders and various cancers.

PRIORITY APPLICATION

This patent application claims priority from pending U.S. provisionalpatent application Ser. No. 62/041,020 filed 22 Aug. 2015.

TECHNICAL FIELD

The present disclosure provides compositions and methods relating to orderived from anti-CXCR3 antibodies. More specifically, the presentdisclosure provides human antibodies that bind CXCR3, CXCR3-bindingfragments and derivatives of such fragments, and CXCR3-bindingpolypeptides comprising such fragments. Further still, the presentdisclosure provides nucleic acids encoding such antibodies, antibodyfragments and derivatives and polypeptides, cells comprising suchpolynucleotides, methods of making such antibodies, antibody fragmentsand derivatives and polypeptides, and methods of using such antibodies,antibody fragments and derivatives and polypeptides, including methodsof treating or diagnosing subjects having CXCR3 related disorders orconditions, including various inflammatory disorders and variouscancers.

BACKGROUND

C—X—C chemokine receptor-3 (CXCR3) is expressed on certain leukocytes,such as activated T cells and NK cells. The CXCR3 receptor bindsligands, such as Interferon Gamma-inducible 10 kD Protein (IP-10),Monokine Induced by Gamma interferon (MIG; Mig), Interferon-inducibleT-cell Alpha Chemoattractant (I-TAC) and B cell-attracting chemokine-1(BCA-1). Certain forms of CXCR3 also bind platelet factor-4 (PF-4,Lasagni et al., J. Exp. Med. 197:1537-1549, 2003). The expression ofsome of the CXCR3 ligands (IP-10, MIG and I-TAC), is induced in tissuesby inteferons or Tumor Necrosis Factor (TNF), potent mediators ofinflammation (Farber, J. M. J. Leukoc. Biol. 61:246-257, 2007; Piali, etal. Eur. J. Immunol. 28:961-972, 1998; and Cole et al. J. Exp. Med.187:2009-2021, 1998). Because of these findings, it has been postulatedthat during inflammation, expression of ligands for CXCR3 isupregulated, resulting in recruitment of CXCR3+ lymphocytes into theinflamed tissue. The infiltrating CXCR3+ lymphocytes can contribute toadverse pathological effects of inflammation. Inhibiting the activitiesof CXCR3, therefore, can have beneficial anti-inflammatory effects.Therefore, there is a need for therapeutic agents that inhibit CXCR3function.

The interaction between chemokines and their receptors is an importantstep in the control of leukocyte migration. Chemokines also mediate avariety of effects independent of chemotaxis, including induction andenhancement of cell-associated cytokine responses.

The human cell surface protein CD183 is a G protein-coupled receptorwith selectivity for three chemokines including IP10(interferon-γ-inducible 10 kDa protein), Mig (monokine induced byinterferon-γ) and I-TAC (interferon-inducible T cell α-chemoattractant).These three chemokines belong to the structural subfamily of “CXC”chemokines, in which a single amino acid residue separates the first twoof four highly conserved Cys residues. Historically, CD183 is the thirdCXC chemokine receptor discovered and, therefore, CD183 is commonlydesignated as “CXCR3.” Binding of chemokines to CXCR3 induces cellularresponses that are involved in leukocyte traffic, most notably integrinactivation, cytoskeletal changes and chemotactic migration. CXCR3 isexpressed on effector/memory T cells and/or in T cells present in manytypes of inflamed tissues (e.g., T-helper 1 cells or Th1 cells and CD8⁺Tc1 cells). In addition, IP10, Mig and I-TAC are commonly produced bylocal cells in inflammatory lesions, suggesting that CXCR3 and itschemokines participate in the recruitment of white blood cells to sitesof inflammation. Therefore, CXCR3 is a target for the development ofantibodies and antagonists, which may be used in the treatment anddiagnosis of diverse inflammatory and immune diseases and disorders,such as rheumatoid arthritis, multiple sclerosis, Crohn's disease,inflammatory bowel disease, chronic obstructive pulmonary disease,psoriasis, type 1 diabetes and transplant rejection. Because CXCR3 isexpressed on a subset of B-cell lymphomas, CXCR3 may also be a targetfor treating and diagnosing lymphomas and leukemias.

Therefore, there is a need in the art for an improved anti-CXCR3antibodies that can be used as therapeutic agents.

SUMMARY

The present disclosure provides a fully human antibody of an IgG classthat binds to an CXCR3 epitope with a binding affinity of at least10⁻⁶M, which has a heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ IDNO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof.Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called 32D12 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQ ID NO. 7/SEQ ID NO. 8(called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called C12 herein), SEQID NO. 11/SEQ ID NO. 12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called E1 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQID NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO. 20(called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11 herein),SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID NO. 25/SEQ IDNO. 26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called 4C3herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO. 31/SEQ ID NO. 33 (calledF7 herein), SEQ ID NO. 31/SEQ ID NO. 34 (called 2A3 herein), SEQ ID NO.31/SEQ ID NO. 35 (called 3A7 herein), SEQ ID NO. 31/SEQ ID NO. 36(called 3A8 herein), SEQ ID NO. 31/SEQ ID NO. 37 (called 4D5 herein),and combinations thereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting SEQ ID NO.1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6,SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQID NO. 37, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has a light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ IDNO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,and combinations thereof. Preferably, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO.24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO.33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO.31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, comprising administering an effectiveamount of an anti-CXCR3 polypeptide, wherein the anti-CXCR3 polypeptideis selected from the group consisting of a fully human antibody of anIgG class that binds to a CXCR3 epitope with a binding affinity of atleast 10⁻⁶M, a fully human antibody Fab fragment, having a variabledomain region from a heavy chain and a variable domain region from alight chain, a single chain human antibody, having a variable domainregion from a heavy chain and a variable domain region from a lightchain and a peptide linker connection the heavy chain and light chainvariable domain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, combinationsthereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has the lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO.22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ IDNO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQID NO. 37, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting SEQ ID NO. 1/SEQ ID NO. 2(called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called 32D12 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQ ID NO. 7/SEQ ID NO. 8(called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called C12 herein), SEQID NO. 11/SEQ ID NO. 12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called E1 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQID NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO. 20(called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11 herein),SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID NO. 25/SEQ IDNO. 26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called 4C3herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO. 31/SEQ ID NO. 33 (calledF7 herein), SEQ ID NO. 31/SEQ ID NO. 34 (called 2A3 herein), SEQ ID NO.31/SEQ ID NO. 35 (called 3A7 herein), SEQ ID NO. 31/SEQ ID NO. 36(called 3A8 herein), SEQ ID NO. 31/SEQ ID NO. 37 (called 4D5 herein),and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4(called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10(called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D12 herein),SEQ ID NO. 13/SEQ ID NO. 14 (called E1 herein), SEQ ID NO. 15/SEQ ID NO.16 (called G12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called 3A3 herein),SEQ ID NO. 19/SEQ ID NO. 20 (called 3A5 herein), SEQ ID NO. 21/SEQ IDNO. 22 (called 3A11 herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12herein), SEQ ID NO. 25/SEQ ID NO. 26 (called 3C11 herein), SEQ ID NO.27/SEQ ID NO. 28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30(called B12 herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQID NO. 31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7 herein),SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID NO. 31/SEQ IDNO. 37 (called 4D5 herein), and combinations thereof. Preferably, thefully human single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO.37, and combinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated thecancer is a CXCR3-positive cancer. Preferably, the broad spectrum ofmammalian cancers to be treated is selected from the group consisting ofleukemias, lymphomas, cancinomas, prostate cancer, non-small cell lungcancer, breast cancer, endometrial cancer, ovarian cancer, gastriccancers, head and neck cancers, melanoma, osteosarcoma, intestinal andcolon cancer, and various metastatic cancers.

The present disclosure further provides a method for treating aninflammatory disorders, comprising administering an effective amount ofan anti-CXCR3 polypeptide, wherein the anti-CXCR3 polypeptide isselected from the group consisting of a fully human antibody of an IgGclass that binds to a CXCR3 epitope with a binding affinity of at least10⁻⁶M, a fully human antibody Fab fragment, having a variable domainregion from a heavy chain and a variable domain region from a lightchain, a single chain human antibody, having a variable domain regionfrom a heavy chain and a variable domain region from a light chain and apeptide linker connection the heavy chain and light chain variabledomain regions, and combinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, andcombinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has the lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO.22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ IDNO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQID NO. 37, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting SEQ ID NO. 1/SEQ ID NO. 2(called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called 32D12 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQ ID NO. 7/SEQ ID NO. 8(called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called C12 herein), SEQID NO. 11/SEQ ID NO. 12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called E1 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQID NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO. 20(called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11 herein),SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID NO. 25/SEQ IDNO. 26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called 4C3herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO. 31/SEQ ID NO. 33 (calledF7 herein), SEQ ID NO. 31/SEQ ID NO. 34 (called 2A3 herein), SEQ ID NO.31/SEQ ID NO. 35 (called 3A7 herein), SEQ ID NO. 31/SEQ ID NO. 36(called 3A8 herein), SEQ ID NO. 31/SEQ ID NO. 37 (called 4D5 herein),and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4(called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10(called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D12 herein),SEQ ID NO. 13/SEQ ID NO. 14 (called E1 herein), SEQ ID NO. 15/SEQ ID NO.16 (called G12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called 3A3 herein),SEQ ID NO. 19/SEQ ID NO. 20 (called 3A5 herein), SEQ ID NO. 21/SEQ IDNO. 22 (called 3A11 herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12herein), SEQ ID NO. 25/SEQ ID NO. 26 (called 3C11 herein), SEQ ID NO.27/SEQ ID NO. 28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30(called B12 herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQID NO. 31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7 herein),SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID NO. 31/SEQ IDNO. 37 (called 4D5 herein), and combinations thereof. Preferably, thefully human single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO.37, and combinations thereof.

Preferably, the inflammatory disorder to be treated is selected from thegroup consisting of rheumatoid arthritis, multiple sclerosis, Crohn'sdisease, Graves disease, Sjögren's syndrome, inflammatory bowel disease,chronic obstructive pulmonary disease, psoriasis, type 1 diabetes,transplant rejection chronic hepatitis C, malaria, and atherosclerosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows binding of the anti-CXCR3 antibody 32B12 to CHO-CXCR3cells.

DETAILED DESCRIPTION

The present disclosure provides a fully human antibody of an IgG classthat binds to a CXCR3 epitope with a binding affinity of 10⁻⁶M or less,that has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and thathas a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35,SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof. Preferably, thefully human antibody has both a heavy chain and a light chain whereinthe antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called 32D12 herein), SEQ IDNO. 5/SEQ ID NO. 6 (called H1 herein), SEQ ID NO. 7/SEQ ID NO. 8 (calledB7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO.11/SEQ ID NO. 12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14(called E1 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQID NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO. 20(called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11 herein),SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID NO. 25/SEQ IDNO. 26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called 4C3herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12 herein), SEQ ID NO.31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO. 31/SEQ ID NO. 33 (calledF7 herein), SEQ ID NO. 31/SEQ ID NO. 34 (called 2A3 herein), SEQ ID NO.31/SEQ ID NO. 35 (called 3A7 herein), SEQ ID NO. 31/SEQ ID NO. 36(called 3A8 herein), SEQ ID NO. 31/SEQ ID NO. 37 (called 4D5 herein),and combinations thereof.

The present disclosure provides a fully human antibody Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO.34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO.31/SEQ ID NO. 37, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has a light chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ IDNO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,and combinations thereof. Preferably, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO.24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO.33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO.31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, and combinations thereof.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers or inflammatory diseases or autoimmunediseases, comprising administering an effective amount of an anti-CXCR3polypeptide, wherein the anti-CXCR3 polypeptide is selected from thegroup consisting of a fully human antibody of an IgG class that binds toa CXCR3 epitope with a binding affinity of at least 10⁻⁶M, a fully humanantibody Fab fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, a single chainhuman antibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, andcombinations thereof;

wherein the fully human antibody Fab fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has the lightchain variable domain sequence that is at least 95% identical to theamino acid sequences selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO.22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ IDNO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQID NO. 37, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO.37, and combinations thereof. Preferably, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ IDNO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO.34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO.31/SEQ ID NO. 37, and combinations thereof. Preferably, the fully humansingle chain antibody has both a heavy chain variable domain region anda light chain variable domain region, wherein the single chain fullyhuman antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ IDNO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO.8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO.13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO.23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO.28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO.35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, andcombinations thereof.

Preferably, the broad spectrum of mammalian cancers to be treated thecancer is a CXCR3-positive cancer. Preferably, the broad spectrum ofmammalian cancers to be treated is selected from the group consisting ofleukemias, lymphomas, cancinomas, prostate cancer, non-small cell lungcancer, breast cancer, endometrial cancer, ovarian cancer, gastriccancers, head and neck cancers, melanoma, osteosarcoma, intestinal andcolon cancer, and various metastatic cancers. Preferably, theinflammatory disorder to be treated is selected from the groupconsisting of rheumatoid arthritis, multiple sclerosis, Crohn's disease,Graves' disease, Sjögren's syndrome, inflammatory bowel disease, chronicobstructive pulmonary disease, psoriasis, type 1 diabetes, transplantrejection chronic hepatitis C, malaria, and atherosclerosis.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (international ImMunoGeneTics information system; Lefrancet al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger andPluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecfic antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US PatentApplications 2002/02512; 2004/0202995; 2004/0038291; 2004/0009507;2003/0039958, and Ward et al., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (Bird et al., 1988, Science242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (Holliger etal., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993 and Poljak et al.,Structure 2:1121-23, 1994). If the two polypeptide chains of a diabodyare identical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra; Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-CXCR3 antibody. In another embodiment, all of the CDRsare derived from a human anti-CXCR3 antibody. In another embodiment, theCDRs from more than one human anti-CXCR3 antibodies are mixed andmatched in a chimeric antibody. For instance, a chimeric antibody maycomprise a CDR1 from the light chain of a first human anti-PAR-2antibody, a CDR2 and a CDR3 from the light chain of a second humananti-CXCR3 antibody, and the CDRs from the heavy chain from a thirdanti-CXCR3 antibody. Other combinations are possible.

Further, the framework regions may be derived from one of the sameanti-CXCR3 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind CXCR3).

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the activation of CXCR3 when an excess of the anti-CXCR3antibody reduces the amount of activation by at least about 20% using anassay such as those described herein in the Examples. In variousembodiments, the antigen binding protein reduces the amount of amount ofactivation of CXCR3 by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, Bowie et al., 1991,Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human CXCR3) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293,293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Preferably, the mammalian cancer to be treated is selected from thegroup consisting of ovarian, colon, breast or hepatic carcinoma celllines, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemia's, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, mast cell derived tumors, andcombinations thereof.

Polypeptides of the present disclosure can be produced using anystandard methods known in the art. In one example, the polypeptides areproduced by recombinant DNA methods by inserting a nucleic acid sequence(e.g., a cDNA) encoding the polypeptide into a recombinant expressionvector and expressing the DNA sequence under conditions promotingexpression.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr.Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 200112(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; andSharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N. Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

CXCR3-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

In one specific embodiment, modified forms of the subject solublepolypeptides comprise linking the subject soluble polypeptides tononproteinaceous polymers. In one specific embodiment, the polymer ispolyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. Examples of the modifiedpolypeptide include PEGylated antibodies.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)-1CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

Although PEG is well-known, this is the first demonstration that apegylated^(10F)n3 polypeptide can be pegylated and retain ligand bindingactivity. In a preferred embodiment, the pegylated^(10F)n3 polypeptideis produced by site-directed pegylation, particularly by conjugation ofPEG to a cysteine moiety at the N- or C-terminus. Accordingly, thepresent disclosure provides a target-binding ^(10F)n3 polypeptide withimproved pharmacokinetic properties, the polypeptide comprising: a^(10F)n3 domain having from about 80 to about 150 amino acids, whereinat least one of the loops of said ^(10F)n3 domain participate in targetbinding; and a covalently bound PEG moiety, wherein said ^(10F)n3polypeptide binds to the target with a K_(D) of less than 100 nM and hasa clearance rate of less than 30 mL/hr/kg in a mammal. The PEG moietymay be attached to the ^(10F)n3 polypeptide by site directed pegylation,such as by attachment to a Cys residue, where the Cys residue may bepositioned at the N-terminus of the ^(0F)n3 polypeptide or between theN-terminus and the most N-terminal beta or beta-like strand or at theC-terminus of the ^(10F)n3 polypeptide or between the C-terminus and themost C-terminal beta or beta-like strand. A Cys residue may be situatedat other positions as well, particularly any of the loops that do notparticipate in target binding. A PEG moiety may also be attached byother chemistry, including by conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem.,252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to CXCR3-binding polypeptides. The number of repeating units“n” in the PEG is approximated for the molecular mass described inDaltons. It is preferred that the combined molecular mass of PEG on anactivated linker is suitable for pharmaceutical use. Thus, in oneembodiment, the molecular mass of the PEG molecules does not exceed100,000 Da. For example, if three PEG molecules are attached to alinker, where each PEG molecule has the same molecular mass of 12,000 Da(each n is about 270), then the total molecular mass of PEG on thelinker is about 36,000 Da (total n is about 820). The molecular massesof the PEG attached to the linker can also be different, e.g., of threemolecules on a linker two PEG molecules can be 5,000 Da each (each n isabout 110) and one PEG molecule can be 12,000 Da (n is about 270).

In a specific embodiment of the disclosure an CXCR3 binding polypeptideis covalently linked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide's6-amino group of a lysine is the available (free) amino group.

The above conjugates may be more specifically presented by formula (II):P—NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR (II), wherein P is the group of abinding polypeptide as described herein, (i.e. without the amino groupor amino groups which form an amide linkage with the carbonyl shown informula (II); and wherein R is lower alkyl; x is 2 or 3; m is from about450 to about 950 and is chosen so that the molecular weight of theconjugate minus the binding polypeptide is from about 10 to about 40kDa. As used herein, the given ranges of “m” have an orientationalmeaning. The ranges of “m” are determined in any case, and exactly, bythe molecular weight of the PEG group.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated binding polypeptide will be usedtherapeutically, the desired dosage, circulation time, resistance toproteolysis, immunogenicity, and other considerations. For a discussionof PEG and its use to enhance the properties of proteins, see Katre,Advanced Drug Delivery Reviews 10: 91-114 (1993).

In one embodiment, PEG molecules may be activated to react with aminogroups on a binding polypeptide, such as with lysines (Bencham et al.,Anal. Biochem., 131, 25 (1983); Veronese et al., Appl. Biochem., 11, 141(1985).; Zalipsky et al., Polymeric Drugs and Drug Delivery Systems,adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky et al., Europ.Polym. J., 19, 1177-1183 (1983); Delgado et al., Biotechnology andApplied Biochemistry, 12, 119-128 (1990)).

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similartype of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of a ^(10F)n3 polypeptide can be performed according to themethods of the state of the art, for example by reaction of the bindingpolypeptide with electrophilically active PEGs (supplier: ShearwaterCorp., USA, www.shearwatercorp.com). Preferred PEG reagents of thepresent invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini et al., BioconjugateChem. 6 (1995) 62-69). Such methods may be used to pegylated at anf-amino group of a binding polypeptide lysine or the N-terminal aminogroup of the binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore et al., Appl. Biochem. Biotechnol.,27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368 (1996);Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897).U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEGspecies that may be coupled to sulfhydryl groups.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into a binding polypeptide coding sequence to generatecysteine residues. This might be achieved, for example, by mutating oneor more amino acid residues to cysteine. Preferred amino acids formutating to a cysteine residue include serine, threonine, alanine andother hydrophilic residues. Preferably, the residue to be mutated tocysteine is a surface-exposed residue. Algorithms are well-known in theart for predicting surface accessibility of residues based on primarysequence or a protein. Alternatively, surface residues may be predictedby comparing the amino acid sequences of binding polypeptides, giventhat the crystal structure of the framework based on which bindingpolypeptides are designed and evolved has been solved (see Himanen etal., Nature. (2001) 20-27; 414(6866):933-8) and thus the surface-exposedresidues identified. In one embodiment, cysteine residues are introducedinto binding polypeptides at or near the N- and/or C-terminus, or withinloop regions.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297, 1059, and U.S. Pat. No. 5,824,784.The use of a PEG-aldehyde for the reductive amination of a proteinutilizing other available nucleophilic amino groups is described in U.S.Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579,and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Patent Publication 2002/0044921 and in WO094/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., Bioconjug. Chem. 2004; 15(5):1005-1009.

Monopegylation of a binding polypeptide can also be produced accordingto the general methods described in WO 94/01451. WO 94/01451 describes amethod for preparing a recombinant polypeptide with a modified terminalamino acid alpha-carbon reactive group. The steps of the method involveforming the recombinant polypeptide and protecting it with one or morebiologically added protecting groups at the N-terminal alpha-amine andC-terminal alpha-carboxyl. The polypeptide can then be reacted withchemical protecting agents to selectively protect reactive side chaingroups and thereby prevent side chain groups from being modified. Thepolypeptide is then cleaved with a cleavage reagent specific for thebiological protecting group to form an unprotected terminal amino acidalpha-carbon reactive group. The unprotected terminal amino acidalpha-carbon reactive group is modified with a chemical modifying agent.The side chain protected terminally modified single copy polypeptide isthen deprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of a binding polypeptide to activated PEG in the conjugationreaction can be from about 1:0.5 to 1:50, between from about 1:1 to1:30, or from about 1:5 to 1:15. Various aqueous buffers can be used inthe present method to catalyze the covalent addition of PEG to thebinding polypeptide. In one embodiment, the pH of a buffer used is fromabout 7.0 to 9.0. In another embodiment, the pH is in a slightly basicrange, e.g., from about 7.5 to 8.5. Buffers having a pKa close toneutral pH range may be used, e.g., phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g. gel filtration) and ion exchange chromatography. Products may alsobe separated using SDS-PAGE. Products that may be separated includemono-, di-, tri-poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety-percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment, PEGylated binding polypeptide of the inventioncontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin PEG-binding polypeptide is from about 3,000 Da to 60,000 Da,optionally from about 10,000 Da to 36,000 Da. In a one embodiment, thePEG in pegylated binding polypeptide is a substantially linear,straight-chain PEG.

In one embodiment of the invention, the PEG in pegylated bindingpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated binding polypeptides of theinvention will preferably retain at least 25%, 50%, 60%, 70%, 80%, 85%,90%, 95% or 100% of the biological activity associated with theunmodified protein. In one embodiment, biological activity refers to itsability to bind to CXCR3, as assessed by KD, k_(on) or k_(off). In onespecific embodiment, the pegylated binding polypeptide protein shows anincrease in binding to CXCR3 relative to unpegylated bindingpolypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Formulations and Modes of Administration

The present disclosure features methods for treating conditions orpreventing pre-conditions which respond to an inhibition of CXCR3biological activity. Preferred examples are conditions that arecharacterized by inflammation or cellular hyperproliferation. Techniquesand dosages for administration vary depending on the type of specificpolypeptide and the specific condition being treated but can be readilydetermined by the skilled artisan. In general, regulatory agenciesrequire that a protein reagent to be used as a therapeutic is formulatedso as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

Exemplary Uses

The CXCR3 binding proteins described herein and their related variantsare useful in a number of therapeutic and diagnostic applications. Theseinclude the inhibition of the biological activity of CXCR3 by competingfor or blocking the binding to a CXCR3 as well as the delivery ofcytotoxic or imaging moieties to cells, preferably cells expressingCXCR3. The small size and stable structure of these molecules can beparticularly valuable with respect to manufacturing of the drug, rapidclearance from the body for certain applications where rapid clearanceis desired or formulation into novel delivery systems that are suitableor improved using a molecule with such characteristics.

On the basis of their efficacy as inhibitors of CXCR3 biologicalactivity, the polypeptides of this disclosure are effective against anumber of cancer conditions as well as complications arising fromcancer, such as pleural effusion and ascites, colorectal cancers,leukemias, lymphomas, head and neck cancers, small cell lung cancer,non-small cell lung cancer (NSCLC) and pancreatic cancer. Non-limitingexamples of cancers include bladder, blood, bone, brain, breast,cartilage, colon kidney, liver, lung, lymph node, nervous tissue, ovary,pancreatic, prostate, skeletal muscle, skin, spinal cord, spleen,stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter,urethra, uterus, or vaginal cancer.

A CXCR3 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Preferably,in breast cancer the combination is with Herceptin or other Her-2therapies.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject anti-CXCR3 antibodies agents of theinvention can be used alone. Alternatively, the subject agents may beused in combination with other conventional anti-cancer therapeuticapproaches directed to treatment or prevention of proliferativedisorders (e.g., tumor). For example, such methods can be used inprophylactic cancer prevention, prevention of cancer recurrence andmetastases after surgery, and as an adjuvant of other conventionalcancer therapy. The present disclosure recognizes that the effectivenessof conventional cancer therapies (e.g., chemotherapy, radiation therapy,phototherapy, immunotherapy, and surgery) can be enhanced through theuse of a subject polypeptide therapeutic agent.

A wide array of conventional compounds has been shown to haveanti-neoplastic activities. These compounds have been used aspharmaceutical agents in chemotherapy to shrink solid tumors, preventmetastases and further growth, or decrease the number of malignant cellsin leukemic or bone marrow malignancies. Although chemotherapy has beeneffective in treating various types of malignancies, manyanti-neoplastic compounds induce undesirable side effects. It has beenshown that when two or more different treatments are combined, thetreatments may work synergistically and allow reduction of dosage ofeach of the treatments, thereby reducing the detrimental side effectsexerted by each compound at higher dosages. In other instances,malignancies that are refractory to a treatment may respond to acombination therapy of two or more different treatments.

When a polypeptide therapeutic agent of the present invention isadministered in combination with another conventional anti-neoplasticagent, either concomitantly or sequentially, such therapeutic agent maybe found to enhance the therapeutic effect of the anti-neoplastic agentor overcome cellular resistance to such anti-neoplastic agent. Thisallows decrease of dosage of an anti-neoplastic agent, thereby reducingthe undesirable side effects, or restores the effectiveness of ananti-neoplastic agent in resistant cells.

Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

Certain chemotherapeutic anti-tumor compounds may be categorized bytheir mechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes—dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (e.g., VEGF inhibitors, fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

Depending on the nature of the combinatory therapy, administration ofthe polypeptide therapeutic agents may be continued while the othertherapy is being administered and/or thereafter. Administration of thepolypeptide therapeutic agents may be made in a single dose, or inmultiple doses. In some instances, administration of the polypeptidetherapeutic agents is commenced at least several days prior to theconventional therapy, while in other instances, administration is beguneither immediately before or at the time of the administration of theconventional therapy.

In one example of a diagnostic application, a biological sample, such asserum or a tissue biopsy, from a patient suspected of having a conditioncharacterized by inappropriate angiogenesis is contacted with adetectably labeled polypeptide of the disclosure to detect levels ofCXCR3. The levels of CXCR3 detected are then compared to levels of CXCR3detected in a normal sample also contacted with the labeled polypeptide.An increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%in the levels of the CXCR3 may be considered a diagnostic indicator.

In certain embodiments, the CXCR3 binding polypeptides are furtherattached to a label that is able to be detected (e.g., the label can bea radioisotope, fluorescent compound, enzyme or enzyme co-factor). Theactive moiety may be a radioactive agent, such as: radioactive heavymetals such as iron chelates, radioactive chelates of gadolinium ormanganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I, or⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imagingagent and is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.Immunoscintigraphy using CXCR3 binding polypeptides directed at CXCR3may be used to detect and/or diagnose cancers and vasculature. Forexample, any of the binding polypeptide against a CXCR3 marker labeledwith ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectively used forsuch imaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,compositions according to the present invention useful as imaging agentscomprising a targeting moiety conjugated to a radioactive moietycomprise 0.1-100 millicuries, in some embodiments preferably 1-10millicuries, in some embodiments preferably 2-5 millicuries, in someembodiments more preferably 1-5 millicuries.

The CXCR3 binding polypeptides can also be used to deliver additionaltherapeutic agents (including but not limited to drug compounds,chemotherapeutic compounds, and radiotherapeutic compounds) to a cell ortissue expressing CXCR3. In one example, the CXCR3 binding polypeptideis fused to a chemotherapeutic agent for targeted delivery of thechemotherapeutic agent to a tumor cell or tissue expressing CXCR3.

The CXCR3 binding polypeptides are useful in a variety of applications,including research, diagnostic and therapeutic applications. Forinstance, they can be used to isolate and/or purify receptor or portionsthereof, and to study receptor structure (e.g., conformation) andfunction.

In certain aspects, the various binding polypeptides can be used todetect or measure the expression of CXCR3, for example, on endothelialcells (e.g., venous endothelial cells), or on cells transfected with aCXCR3 gene. Thus, they also have utility in applications such as cellsorting and imaging (e.g., flow cytometry, and fluorescence activatedcell sorting), for diagnostic or research purposes.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to CXCR3. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a CXCR3protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the CXCR3 protein. In oneembodiment, a sample containing cells expressing a CXCR3 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a CXCR3protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of a CXCR3protein in a biological sample can also be prepared. Such kits willinclude a CXCR3 binding polypeptide which binds to a CXCR3 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Similarly, the present disclosure also provides a method of detectingand/or quantitating expression of CXCR3, wherein a compositioncomprising a cell or fraction thereof (e.g., membrane fraction) iscontacted with a binding polypeptide which binds to a CXCR3 or portionof the receptor under conditions appropriate for binding thereto, andthe binding is monitored. Detection of the binding polypeptide,indicative of the formation of a complex between binding polypeptide andCXCR3 or a portion thereof, indicates the presence of the receptor.Binding of a polypeptide to the cell can be determined by standardmethods, such as those described in the working examples. The method canbe used to detect expression of CXCR3 on cells from an individual.Optionally, a quantitative expression of CXCR3 on the surface ofendothelial cells can be evaluated, for instance, by flow cytometry, andthe staining intensity can be correlated with disease susceptibility,progression or risk.

The present disclosure also provides a method of detecting thesusceptibility of a mammal to certain diseases. To illustrate, themethod can be used to detect the susceptibility of a mammal to diseaseswhich progress based on the amount of CXCR3 present on cells and/or thenumber of CXCR3-positive cells in a mammal.

Polypeptide sequences are indicated using standard one- or three-letterabbreviations. Unless otherwise indicated, each polypeptide sequence hasamino termini at the left and a carboxy termini at the right; eachsingle-stranded nucleic acid sequence, and the top strand of eachdouble-stranded nucleic acid sequence, has a 5′ termini at the left anda 3′ termini at the right. A particular polypeptide sequence also can bedescribed by explaining how it differs from a reference sequence.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The terms “CXCR3 inhibitor” and “CXCR3 antagonist” are usedinterchangeably. Each is a molecule that detectably inhibits at leastone function of CXCR3. Conversely, an “CXCR3 agonist” is a molecule thatdetectably increases at least one function of CXCR3. The inhibitioncaused by an CXCR3 inhibitor need not be complete so long as it isdetectable using an assay. Any assay of a function of CXCR3 can be used,examples of which are provided herein. Examples of functions of CXCR3that can be inhibited by a CXCR3 inhibitor, or increased by aCXCR3agonist, include cancer cell growth or apoptosis (programmed celldeath), and so on. Examples of types of CXCR3 inhibitors and CXCR3agonists include, but are not limited to, CXCR3 binding polypeptidessuch as antigen binding proteins (e.g., CXCR3 inhibiting antigen bindingproteins), antibodies, antibody fragments, and antibody derivatives.

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Preferably, the anti-CXCR3 antibodies disclosed herein are characterizedby their variable domain region sequences in the heavy V_(H) and lightV_(L) amino acid sequences. The preferred antibody is A6 which is akappa IgG antibody. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)). The variable regions of eachlight/heavy chain pair form the antibody binding site such that anintact immunoglobulin has two binding sites.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human CXCR3) if it binds to the antigen with a dissociation constant of1 nanomolar or less.

An “antigen binding domain, “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent homology” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

A “host cell” is a cell that can be used to express a nucleic acid. Ahost cell can be a prokaryote, for example, E. coli, or it can be aeukaryote, for example, a single-celled eukaryote (e.g., a yeast orother fungus), a plant cell (e.g., a tobacco or tomato plant cell), ananimal cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell) or a hybridoma. Examples of hostcells include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivativessuch as Veggie CHO and related cell lines which grow in serum-free media(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11,which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci.USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonickidney cells such as 293,293 EBNA or MSR 293, human epidermal A431cells, human Colo205 cells, other transformed primate cell lines, normaldiploid cells, cell strains derived from in vitro culture of primarytissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, ahost cell is a cultured cell that can be transformed or transfected witha polypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

Antigen binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to CXCR3, (preferably, human CXCR3). Antigen binding proteinsinclude antigen binding proteins that inhibit a biological activity ofCXCR3.

Oligomers that contain one or more antigen binding proteins may beemployed as CXCR3 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have CXCR3 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a CXCR3 binding fragment of an anti-CXCR3 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-CXCR3 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-CXCR3 antibody fragments or derivatives thatform are recovered from the culture supernatant.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments.

The present disclosure provides monoclonal antibodies that bind toCXCR3. Monoclonal antibodies may be produced using any technique knownin the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Antigen binding proteins directed against CXCR3 can be used, forexample, in assays to detect the presence of CXCR3 polypeptides, eitherin vitro or in vivo. The antigen binding proteins also may be employedin purifying CXCR3 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as CXCR3 antagonists may beemployed in treating any CXCR3-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit CXCR3-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of CXCR3, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of a CXCR3 blockingantigen binding protein to a mammal in need thereof in an amounteffective for reducing a CXCR3-induced biological activity.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of CXCR3.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N. Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of CXCR3bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-CXCR3 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-CXCR3 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP->CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for CXCR3 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from CXCR3. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to ⁻¹ orlower. In another embodiment, the K_(off) is 5×10⁻⁵ to ⁻¹ or lower. Inanother embodiment, the K_(off) is substantially the same as an antibodydescribed herein. In another embodiment, the antigen binding proteinbinds to CXCR3 with substantially the same K_(off) as an antibodydescribed herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of CXCR3. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of CXCR3with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human CXCR3 expressed on the surface of a celland, when so bound, inhibits CXCR3 signaling activity in the cellwithout causing a significant reduction in the amount of CXCR3 on thesurface of the cell. Any method for determining or estimating the amountof CXCR3 on the surface and/or in the interior of the cell can be used.In other embodiments, binding of the antigen binding protein to theCXCR3-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface CXCR3 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of CXCR3, or to anepitope of CXCR3 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise an CXCR3 binding site from oneof the herein-described antibodies and a second CXCR3 binding regionfrom another of the herein-described antibodies, including thosedescribed herein by reference to other publications. Alternatively, abispecific antigen binding protein may comprise an antigen binding sitefrom one of the herein described antibodies and a second antigen bindingsite from another CXCR3 antibody that is known in the art, or from anantibody that is prepared by known methods or the methods describedherein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

95% Homology

The present disclosure provides a number of antibodies structurallycharacterized by the amino acid sequences of their variable domainregions. However, the amino acid sequences can undergo some changeswhile retaining their high degree of binding to their specific targets.More specifically, many amino acids in the variable domain region can bechanged with conservative substitutions and it is predictable that thebinding characteristics of the resulting antibody will not differ fromthe binding characteristics of the wild type antibody sequence. Thereare many amino acids in an antibody variable domain that do not directlyinteract with the antigen or impact antigen binding and are not criticalfor determining antibody structure. For example, a predictednonessential amino acid residue in any of the disclosed antibodies ispreferably replaced with another amino acid residue from the same class.Methods of identifying amino acid conservative substitutions which donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al, ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997)). Near et al. Mol. Immunol. 30:369-377, 1993 explainshow to impact or not impact binding through site-directed mutagenesis.Near et al. only mutated residues that they thought had a highprobability of changing antigen binding. Most had a modest or negativeeffect on binding affinity (Near et al. Table 3) and binding todifferent forms of digoxin (Near et al. Table 2).

Indications

In one aspect, the present disclosure provides methods of treating asubject. The method can, for example, have a generally salubrious effecton the subject, e.g., it can increase the subject's expected longevity.Alternatively, the method can, for example, treat, prevent, cure,relieve, or ameliorate (“treat”) a disease, disorder, condition, orillness (“a condition”). Among the conditions to be treated areconditions characterized by inappropriate expression or activity ofCXCR3. In some such conditions, the expression or activity level is toohigh, and the treatment comprises administering a CXCR3 antagonist asdescribed herein. The disorders or conditions are cancer-related. Inparticular, those cancers include, but are not limited to, breast, lung,ovarian and colon carcinoma and various myelomas.

Specific medical conditions and diseases that are treatable orpreventable with the antigen binding proteins of this disclosure includevarious cancers.

Therapeutic Methods and Administration of Antigen Binding Proteins

Certain methods provided herein comprise administering a CXCR3 bindingantigen binding protein to a subject, thereby reducing a CXCR3-inducedbiological response that plays a role in a particular condition. Inparticular embodiments, methods of the invention involve contactingendogenous CXCR3 with an CXCR3 binding antigen binding protein, e.g.,via administration to a subject or in an ex vivo procedure.

The term “treatment” encompasses alleviation or prevention of at leastone symptom or other aspect of a disorder, or reduction of diseaseseverity, and the like. An antigen binding protein need not effect acomplete cure, or eradicate every symptom or manifestation of a disease,to constitute a viable therapeutic agent. As is recognized in thepertinent field, drugs employed as therapeutic agents may reduce theseverity of a given disease state, but need not abolish everymanifestation of the disease to be regarded as useful therapeuticagents. Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition in order toconstitute a viable prophylactic agent. Simply reducing the impact of adisease (for example, by reducing the number or severity of itssymptoms, or by increasing the effectiveness of another treatment, or byproducing another beneficial effect), or reducing the likelihood thatthe disease will occur or worsen in a subject, is sufficient. Oneembodiment of the invention is directed to a method comprisingadministering to a patient a CXCR3 antagonist in an amount and for atime sufficient to induce a sustained improvement over baseline of anindicator that reflects the severity of the particular disorder.

As is understood in the pertinent field, pharmaceutical compositionscomprising the antibodies and fragments thereof of the disclosure areadministered to a subject in a manner appropriate to the indication.Pharmaceutical compositions may be administered by any suitabletechnique, including but not limited to, parenterally, topically, or byinhalation. If injected, the pharmaceutical composition can beadministered, for example, via intra-articular, intravenous,intramuscular, intralesional, intraperitoneal or subcutaneous routes, bybolus injection, or continuous infusion. Localized administration, e.g.at a site of disease or injury is contemplated, as are transdermaldelivery and sustained release from implants. Delivery by inhalationincludes, for example, nasal or oral inhalation, use of a nebulizer,inhalation of the antagonist in aerosol form, and the like. Otheralternatives include eyedrops; oral preparations including pills,syrups, lozenges or chewing gum; and topical preparations such aslotions, gels, sprays, and ointments.

Use of antigen binding proteins in ex vivo procedures also iscontemplated. For example, a patient's blood or other bodily fluid maybe contacted with an antigen binding protein that binds CXCR3 ex vivo.The antigen binding protein may be bound to a suitable insoluble matrixor solid support material.

Advantageously, antigen binding proteins are administered in the form ofa composition comprising one or more additional components such as aphysiologically acceptable carrier, excipient or diluent. Optionally,the composition additionally comprises one or more physiologicallyactive agents, for example, a second inflammation- or immune-inhibitingsubstance, an anti-angiogenic substance, an analgesic substance, etc.,non-exclusive examples of which are provided herein. In variousparticular embodiments, the composition comprises one, two, three, four,five, or six physiologically active agents in addition to an CXCR3binding antigen binding protein

Combination Therapy

In another aspect, the present disclosure provides a method of treatinga subject with an CXCR3 inhibiting antigen binding protein and one ormore other treatments. In one embodiment, such a combination therapyachieves synergy or an additive effect by, for example, attackingmultiple sites or molecular targets in a tumor. Types of combinationtherapies that can be used in connection with the present inventioninclude inhibiting or activating (as appropriate) multiple nodes in asingle disease-related pathway, multiple pathways in a target cell, andmultiple cell types within a target tissue.

In another embodiment, a combination therapy method comprisesadministering to the subject two, three, four, five, six, or more of theCXCR3 agonists or antagonists described herein. In another embodiment,the method comprises administering to the subject two or more treatmentsthat together inhibit or activate (directly or indirectly)CXCR3-mediated signal transduction. Examples of such methods includeusing combinations of two or more CXCR3 inhibiting antigen bindingproteins, of an CXCR3 inhibiting antigen binding protein and one or moreother therapeutic moiety having anti-cancer properties (for example,cytotoxic agents, and/or immunomodulators), or of an CXCR3 inhibitingantigen binding protein and one or more other treatments (e.g., surgery,or radiation). Furthermore, one or more anti-CXCR3 antibodies orantibody derivatives can be used in combination with one or moremolecules or other treatments, wherein the other molecule(s) and/ortreatment(s) do not directly bind to or affect CXCR3, but whichcombination is effective for treating or preventing the condition beingtreated. In one embodiment, one or more of the molecule(s) and/ortreatment(s) treats or prevents a condition that is caused by one ormore of the other molecule(s) or treatment(s) in the course of therapy,e.g., nausea, fatigue, alopecia, cachexia, insomnia, etc. In every casewhere a combination of molecules and/or other treatments is used, theindividual molecule(s) and/or treatment(s) can be administered in anyorder, over any length of time, which is effective, e.g.,simultaneously, consecutively, or alternately. In one embodiment, themethod of treatment comprises completing a first course of treatmentwith one molecule or other treatment before beginning a second course oftreatment. The length of time between the end of the first course oftreatment and beginning of the second course of treatment can be anylength of time that allows the total course of therapy to be effective,e.g., seconds, minutes, hours, days, weeks, months, or even years.

In another embodiment, the method comprises administering one or more ofthe CXCR3 antagonists described herein and one or more other treatments(e.g., a therapeutic or palliative treatment). Where a method comprisesadministering more than one treatment to a subject, it is to beunderstood that the order, timing, number, concentration, and volume ofthe administrations is limited only by the medical requirements andlimitations of the treatment, i.e., two treatments can be administeredto the subject, e.g., simultaneously, consecutively, alternately, oraccording to any other regimen.

Example 1

This example shows a cellular binding assay to determine the specificbinding of anti-CXCR3 antibodies to CXCR3 expressed on a stable cellline. CHO-CXCR3 cells were lifted from culture flasks usingnon-enzymatic Cell Dissociation Buffer—PBS based (Life Technologies#13151-014). Cells were resuspended in FACS Buffer (2% Fetal BovineSerum in PBS) at 1×10⁶ cells/ml and 50 μl (0.5×10⁵ cells) were aliquotedinto the wells of a 96-well plate. At the same time, CHO cellsexpressing an unrelated protein were treated in the same manner.Aliquots of antibody to give two different final concentrations, mostoften 5 and 10 μg/ml, were added to the cells and incubated for 1 hourat 4° C. and then washed twice with FACS Buffer. Cells were resuspendedin 50 μl goat anti-human IgG (γ-chain specific)-PE conjugated secondaryantibody (Southern Biotech #2040-09) diluted 1:500 in FACS Buffer. Cellswere further incubated for 30 min at 4° C. and then washed 1× with FACSBuffer. The cells were resuspended in a final volume of 30 μl FACSBuffer and analyzed using the Intellicyt Flow Cytometer. Medianfluorescence in the FL-2H channel was determined using FlowJo software.Binding of each antibody to the CHO-CXCR3 cell line was compared tobinding to a control CHO cell line. The list of antibodies that showedspecific binding to CHO-CXCR3 cells is shown in Table 1.

TABLE 1 Antibodies that bind to CHO-CXCR3. Name Binding to hu-CXCR3 CHOB2 yes C12 yes F7 yes 2A3 yes 3A3 yes 3A11 yes 3C11 yes 4D5 yes 32B12yes 32D12 yes H1 yes C4 yes 3A7 yes 3A8 yes 3A5 yes

Example 2

This example shows a cellular binding assay to determine the EC₅₀ foranti-CXCR3 antibodies binding to human CXCR3. This example shows thebinding characteristics for these antibodies in terms of the maximalcell binding and the concentration at which 50% binding saturation(EC₅₀) is reached. In this example, CHO-CXCR3 cells were lifted fromculture flasks using non-enzymatic Cell Dissociation Buffer—PBS based(Life Technologies #13151-014). Cells were resuspended in FACS Buffer(2% Fetal Bovine Serum in PBS) at 1×10⁶ cells/ml and 50 μl (0.5×10⁵cells) were aliquoted into the wells of a 96-well plate. Plated cellswere spun down and the supernatant discarded. Cells were resuspended in30 μl FACS Buffer containing serially diluted concentrations of antibodyin triplicate. Plates were incubated for 1 hr at 4° C. and then washedtwice with FACS Buffer. Cells were resuspended in 50 μl goat anti-humanIgG (γ-chain specific)-PE conjugated secondary antibody (SouthernBiotech #2040-09) diluted 1:500 in FACS Buffer. Cells were furtherincubated for 30 min at 4° C. and then washed 1× with FACS Buffer. Thecells were resuspended in a final volume of 30 μl FACS Buffer andanalyzed using the Intellicyt Flow Cytometer. Median fluorescence in theFL-2H channel was determined using FlowJo software and EC₅₀ value wasdetermined by plotting the data in GraphPad Prism and analyzing using avariable slope non-linear regression. The results are shown in Table 2.

TABLE 2 EC₅₀ of anti-CXCR3 antibodies to human CXCR3. Name EC50 (nM),hu-CXCR3 CHO B2 0.7 C12 15 F7 5 2A3 11 3A3 0.5 3A11 1.2 3C11 0.6 4D5 732B12 1 32D12 4

Example 3

This example shows a cellular binding assay to determine the EC₅₀ foranti-CXCR3 antibodies binding to murine CXCR3. This example shows thebinding characteristics for these antibodies in terms of the maximalcell binding and the concentration at which 50% binding saturation(EC₅₀) is reached. In this example, murine B16-F10 cells (ATCC,#CRL-6475) were lifted from culture flasks using non-enzymatic CellDissociation Buffer—PBS based (Life Technologies #13151-014). Cells wereharvested, washed and resuspended in FACS Buffer (2% Fetal Bovine Serumin PBS) at 1×10⁶ cells/ml and 50 μl (0.5×10⁵ cells) were aliquoted intothe wells of a 96-well plate. Plated cells were spun down and thesupernatant discarded. Cells were resuspended in 30 μl FACS Bufferserially diluted concentrations of antibody in triplicate. Plates wereincubated for 1 hr at 4° C. and then washed 2× with FACS Buffer. Cellswere resuspended in 50 μl goat anti-human IgG (γ-chain specific)-PEconjugated secondary antibody (Southern Biotech #2040-09) diluted 1:500in FACS Buffer. Cells were further incubated for 30 min at 4° C. andthen washed 1× with FACS Buffer. The cells were resuspended in a finalvolume of 30 FACS Buffer and analyzed using the Intellicyt FlowCytometer. Median fluorescence in the FL-2H channel was determined usingFlowJo software and the EC₅₀ value was determined by plotting the datain GraphPad Prism and analyzing using a variable slope non-linearregression. The results are shown in Table 3.

TABLE 3 EC₅₀ of anti-CXCR3 antibodies to murine CXCR3. Name EC50 (nM),mur CXCR3 32B12 25

Example 4

In this example, CHO-CXCR3 cells were lifted from culture flasks usingnon-enzymatic Cell Dissociation Buffer—PBS based (Life Technologies#13151-014). Cells were resuspended in FACS Buffer (2% Fetal BovineSerum in PBS) with sodium azide at 1×10⁶ cells/ml and 800 were aliquotedinto a tube for each antibody. Antibody was added to a finalconcentration of 5 μg/ml and incubated at room temperature for 1 h. Thesamples were placed on ice and a 105 aliquot at various time points wasadded to 190 μl FACS buffer in a 96-well plate and centrifuged for 3min. Supernatant was discarded, the cells were resuspended in 105 μl ofFACS buffer, duplicate aliquots (50 μl) added to another 96-well platecontaining 150 μl FACS buffer and incubated at room temperature. Afterall of the time points were collected, the plate was centrifuged for 3min, supernatant discarded and the cells resuspended in 50 μl goatanti-human IgG (γ-chain specific)-PE conjugated secondary antibody(Southern Biotech #2040-09) diluted 1:500 in FACS Buffer. Cells wereincubated for 30 min at 4° C. and then washed 1× with FACS Buffer. Thecells were resuspended in a final volume of 30 μl FACS Buffer andanalyzed using the Intellicyt Flow Cytometer. The binding of clone 32B12over time is shown in FIG. 1.

Example 5

This example shows a calcium assay using anti-CXCR3 antibodies.CHO-CXCR3 cells (CHO cells transfected with human CXCR3 gene) were usedin calcium assay. Aliquots of 0.1×10⁵ cells were seeded in 20 μl ofgrowth medium per well of a 384 well plate (#781091, Greiner) andincubated for 20 hours. The growth medium was F-12K (#21127, LifeTechnologies) plus 10% FBS. 5 μl of serial diluted antibodies were addedinto 20 μl cells and incubated 20 min at room temperature. Then 25 μl ofCalcium 4 Assay kit (R8142, Molecular Devices) including 0.5 mMprobenecid (Sigma #P8761-25G) were added to the 25 μl cellspre-incubated with the antibodies. After 1 h incubation at 37° C. 5% CO₂followed by 15 minutes at room temperature, 12.5 μl of 5× dose ofligands were added to each well of the plate as the challenge agonistduring detection on the FlexStation3. The final concentration of eachligand used in the assay was 200 nM of CXCL9 (#300-26, PeproTech), 164nM of CXCL10 (#300-12, PeproTech) and 60 nM of CXCL11 (#300-46,PeproTech). The IC₅₀ value of each antibody shown in the table wascalculated using Prism5. The IC₅₀ of each antibody with eachcorresponding ligand is shown in Table 4. Each IC₅₀ value represents theMean (n=2). The control antibody is from R&D (Cat# MAB 160, R&D).

TABLE 4 IC₅₀ [nM] of anti-CXCR3 antibodies in a calcium flux assay usingCHO-CXCR3 cells CXCL9 CXCL10 CXCL11 R&D mAb n.d. n.d. 23 32B12 37 7 1632D12 n.d. n.d. 19 Note: n.d. = not determined

Example 6

This example describes chemotaxis assays using activated human T cells.Primary human T cells activated by anti-CD3 (1 ng/ml) for 2 days wereused for prepared. Chemotaxis assays were set up using a 96-wellchemotaxis chamber (ChemoTX; NeuroProbe, Gaithersburg, Md.) with the 2compartments separated by a 5-μm polycarbonate membrane. The assaybuffer used for chemotaxis was RPMI plus 0.5% BSA. Serially-dilutedconcentrations of antibodies were pre-incubated with the activated Tcells (0.1×10⁶ cells in 25 μl of the assay buffer) at room temperaturefor 20 min. The ligands CXCL9 (30 nM), CXCL10 (12.5 nM) and CXCL11 (0.3nM) were loaded respectively in the wells of plate in the lowerchambers, whereas the cells pre-incubated with antibodies were loaded ontop of membrane. After 2 h incubation at 37° C., 5% CO₂, migrated cellsin the lower chamber were transferred to an opaque white 96-well plateand incubated with CellTiter GLo (Cat#G7571, Promega) for 10 min at roomtemperature. Luminescent signals generated from the cells in each wellwere detected by a fluorescent plate reader (FlexStation3, MolecularDevice). The IC₅₀ value of each mAb indicated in the table wascalculated by Prism5. The IC₅₀ of the each mAb against correspondingligands is indicated in Table 5. Each IC₅₀ value represents Mean (n=2).The control antibody was from R&D (Cat# MAB 160, R&D).

TABLE 5 IC₅₀ [nM] of anti-CXCR3 antibodies in a chemotaxis assay usingactivated T cells CXCL9 CXCL10 CXCL11 R&D mAb 1 1.8 0.4 32B12 0.6 0.20.3 32D12 2.5 0.03 3.4

Sequence Listing Heavy chain Light chain variable domain regionvariable domain region 32B12 EVQLVQSGGGLVKSGRSLRLSCSYELTQPPSVSAAPGQKVTISC TVSGFTFGNSAMSWFRQAPGKG SGSSSNIGNSYVSWFQQFPGTALEWVSSISSSSSYIYYADSVKG PKLVIYENNKRPSGIPDRFSGS RFTISRDNAKNSLYLQMNSLRAKSGTSATLGITGLQTGDEADYY EDTAVYYCARDLSGRGYSYLNY CGTWDSSLSGRVFGGGTKLTVLWGQGTLVTVSS SEQ ID NO. 2 SEQ ID NO. 1 32D12 EVQLLESGGGLVQPGGSQRLSCLPVLTQPASVSGSPGQPITISC AASGFTFSSYPMTWVRQAPGKG TGTSGNIGSYNYVSWYQQHPGKLEWVSAISDGGSGTYYADSVKG APRLLIYDVSLRPSGISSHFSG RFTISRDNSKNTLYLQMNSLRASKSGNTASLTISGLQAEDEAEY EDTAIYYCARYKMGGSYRGLDD FCLSWTSDKTYVFGSGTKLTVLWGQGTLVTVSS SEQ ID NO. 4 SEQ ID NO. 3 H1 EVQLLESGGGVVQPGRSLRLSCQAGLTQPPSVSAAPGQKVTISC AASGFTFRYYGMHWVRQAPGKG SGSSSNIGNNYVSWYQQLPGTALEWVAAIRNDGSNEYYADSVKG PKLLIYDNKRRPSGMPDRFSGS RFTISRDNSKNTLYLEMNSLRAKSGTSATLGITGLQTGDEADYY EDTSVYHCARDGFYRGSPYYFD CGTWDSSLSAVVFGGGTKLTVLSWGQGTLVTVSS SEQ ID NO. 6 SEQ ID NO. 5 B7 QVQLVQSGAEVKKPGSSVKVSCDIVMTQTPLSSPVTLGQPASIS KASGGTFKRYSVHWVRQAPGQG CRSSQSLVHSDGNTYLSWFQQRLEWVGRIVPFLGLANYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTITADTSTNTTYMDLRSLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCVRSTGYLDYWGQGT GVYFCLQSLQLPITFGQGTKVELVTVSS SEQ ID NO. 7 IK SEQ ID NO. 8 C12 QVQLVQSGAEVKKPGSSMKVSCDIVMTQSPLSLPVTLGQPASIS KTSGGTFNAYAITWVRQAPGQG CRSSQSLVYSDGNTYLNWFQQRLEWMGRIVPVLGMVSYVQKFQG PGQSPRRLIYKVSNRESGVPDR RLTITADKSTSTTYMELSSLTSFSGSGSGTDFTLKISRVEAEDV DDTAVYYCARQTGDSDHWGPGT GVYYCMQGTHWPNTFGGXTKLELVTVSS SEQ ID NO. 9 IK SEQ ID NO. 10 D12 QMQLVQSGAEVKKPGASVKVSCQTVVTQPPSVSGAPGQRVTVSC KASGYTFTSYYMHWVRQAPGQG TGSSSNIGSRYAVNWYQQLPGRLEWMGIINPSAGSTSYAQKFQG APKLLIYGNTNRPSGVPDRFSA RVTMTRDTSTSTVYMELSSLGSSKSGTSASLAISGLQAEDEADY EDTAVYYCASRSFGNDGVFDIW YCQSFDSSLRGYVFGTGTKVTVGQGTMVTVSS L SEQ ID NO. 12 SEQ ID NO. 11 E1 EVQLVESGGGVVQPGRSLRLSCSDVVMTQTPSTLSASVGDRVTI AASEFTFSSYTIHWVRQAPGKG TCRASQSIKTWLAWYQQRPGKALEWVGRIHPKSEGGTTDYAAPV PKRLIYAASSLQSGVPSRFSGS KGRFLISRDDSENTMYLQMNSLGSGTEFTLTISSLQPEDFATYY QTEDTAVYYCATYGSGSHWIWG CVQHNSYPITFGQGTRLEIKQGTLVTVSS SEQ ID NO. 14 SEQ ID NO. 13 G12 EVQLVESGGGVVQPGKSLRLSCDIVMTQTPLSLPVTLGQPASLS AASGFTFSSYGMHWVRQAPGKG CRSSQSLAHRDGNTYLSWFQQRLEWVAVISYDGSNKYYADSVKG PGQSPRRLIYKVSNRDSGVPDR RFTISRDNSKNTLYLQMNSLRAFSGSGSGTDFTLKISRVEAEDV EDTAVYYCAYGGFSHWGQGTLV GVYYCMQGTHWPPTFGGGTKVETVSS SEQ ID NO. 15 IK SEQ ID NO. 16 3A3 QVQLVQSGAEVKKPGSSVKVSCDIVMTQTPLSLPVTLGQPASIS KASGGTFNRYAFSWVRQAPGQG CRSSQSLVHNDGNTYLNWFHQRPELMGRIMPMFDIASYAQKFQG PGQSPRRLIYKVSNRDFGVPDR RLTITADKSTSTAYMELSSLRSFSGSGSGTDFTLRISRVEAEDV DDTAVYYCTQVSGDSDYWGPGT GVYYCMQGSHWPLTFGGGTKVELVTVSS SEQ ID NO. 17 IK SEQ ID NO. 18 3A5 QVQLVQSGAEVKKPGSSVKVACDVVMTQTPLSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CRSSQSLVYRDGNTYLNWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPITFGQGTKVETLVTVSS SEQ ID NO 19 IK SEQ ID NO. 20 3A11 QVQLVQSGGGLVKPGGSLRLSCSYVLTQPPSVSKGLRQTATLTC AASGFTFTSSGMNWVRQAPGKG TGNINNVGNVGATWLQQHQGHPLEWVAYIFPNGKTLYPESVKGR PKLLSDRNNNRPSGISERFSAS FIITRDNAEKSLFLEMNNLRAERSGNAASLTITGLQPEDEADYY DMGVYYCAIDLNWGSGYWGQGT CSAWDTSLGAWVFGGGTKLTVLLVTVSS SEQ ID NO 21 SEQ ID NO. 22 3B12 EVQLVESGGGVVQPGRSLRLSCQSVLTQPPSVSKDLRQTATLTC AASGFTFSHYGMHWVRQAPGKG TGNSNNVGDKGAAWLQQHQGHPLEWVAVISYDGSKKSYADSVKG PKLLSYRNNNRPSGISERLSAS RFTISRDNSKNTLYLQMNSLRARSGNTASLTITGLQPDDEADYY DDTAVYYCATSAGGGGYWGQGT CSAWDSSLSAWVFGGGTKLTVLLVTVSS SEQ ID NO 23 SEQ ID NO. 24 3C11 EVQLVESGGGLVQPGGSLRLSCDIQLTQSPSSLSASVGDRVTIT SASGFTFSSYAMHWVRQAPGKG CRASQGIGTYLAWFQQKPGKVPLEYVSAISSNGGSTYYADSVKG KPLIYAASTLQSGVPSRFSGSG RFTISRDNSKNTLYLQMSSLRASGADFTLTISSLQPEDVATYYC EDTAVYYCVKSIAAAGTQYYYY QKYNSVPQTFGQGTKVEIKYMDVWGKGTTVTVSS SEQ ID NO. 26 SEQ ID NO 25 4C3 QVQLVESGGGLVQPGRSLRLSCAIQLTQSPLALPVPLGQPASIS IISGFTFGDYTMSWFRQAPGKG CRASQSLVYSNGNTYLNWFQLRLEWVGFIRSKAYGGTTQYAASV PGQSPRRLIYKVSRRDSGVPDR KGRFTISRDDSKSIAYLQMNSLFSGSGSDTDFTLKISRVEAEDV KTEDTAVYYCTRGDYGDFPWYF GVYYCMQATQWPITFGQGTRLEDLWGRGTLVTVSS IK SEQ ID NO. 28 SEQ ID NO 27 B12 QMQLVQSGAEVKKPGSSVKVSCDVVMTQSPLSLPVTLGQPASIS KASGGTFNRYAFSWVRQAPGQG CRSTESLVHSDGNTYLNWFHQRPELMGRIMPMFDIASYAQKFQG PGQSPRRLIYKISNRDSGVPDR RLTITADKSTSTAYMELSSLRSFSGSGSGTDFTLKISRVEAEDV DDTAVYYCTQVSGDSDYWGPGT GVYYCFQGTHWPPTFGQGTKVETVTVSS SEQ ID NO. 29 IK SEQ ID NO. 30 C4 QVQLVQSGAEVKKPGSSVKVACEIVLTQSPLSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CNSSQSLLHRDGHTYLHWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDK RVTISADKSTRTGYMELRDLRSFSGSGSGTEFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPLAFGGGTKLETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 32 F7 QVQLVQSGAEVKKPGSSVKVACDIVMTQTPLSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CRSNQSLVHSDGNTYLYWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKINRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPNTFGQGTKLETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 33 2A3 QVQLVQSGAEVKKPGSSVKVACDIVMTQTPLSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CRSSQSLVHSDGNTYLNWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPLTFGGGTKVETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 34 3A7 QVQLVQSGAEVKKPGSSVKVACDVVMTQSPVSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CRSSQSLVHSDGNTYFNWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPITFGQGTRLETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 35 3A8 QVQLVQSGAEVKKPGSSVKVACDIVMTQTPLSLPVTLGQPAAIS KASGGTFSHSAISWVRQAPGQG CRSSQSLVYSDGNTYLNWFHQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPLTFGGGTKLETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 36 4D5 QVQLVQSGAEVKKPGSSVKVACDIVMTQSPLSLPVTLGQPASIS KASGGTFSHSAISWVRQAPGQG CRSSHTLVHSDGTTYLNWFQQRLEWIGRTIPTLEMASYAQKFQG PGQSPRRLIYKVSNRDSGVPDR RVTISADKSTRTGYMELRDLRSFSGSGSGTDFTLKISRVEAEDV EDTAVYYCSTQTPSYTDHWGQG GVYYCMQGTHWPLTFGGGTKVETLVTVSS SEQ ID NO. 31 IK SEQ ID NO. 37

We claim:
 1. A fully human antibody of an IgG class that binds to aCXCR3 epitope with a binding affinity of at least 10⁻⁶M, that has aheavy chain variable domain sequence that is at least 95% identical tothe amino acid sequences selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and that has alight chain variable domain sequence that is at least 95% identical tothe amino acid sequences selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 37, and combinations thereof.
 2. The fully human antibodyof claim 1, wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called 32D12herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1 herein), SEQ ID NO. 7/SEQID NO. 8 (called B7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called C12herein), SEQ ID NO. 11/SEQ ID NO. 12 (called D12 herein), SEQ ID NO.13/SEQ ID NO. 14 (called E1 herein), SEQ ID NO. 15/SEQ ID NO. 16 (calledG12 herein), SEQ ID NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO.19/SEQ ID NO. 20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22(called 3A11 herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein),SEQ ID NO. 25/SEQ ID NO. 26 (called 3C11 herein), SEQ ID NO. 27/SEQ IDNO. 28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34 (called2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7 herein), SEQ ID NO.31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID NO. 31/SEQ ID NO. 37(called 4D5 herein), and combinations thereof.
 3. A fully human antibodyFab fragment, having a variable domain region from a heavy chain and avariable domain region from a light chain, wherein the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.
 4. The fully human antibody Fabfragment of claim 3, wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO.34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO.31/SEQ ID NO. 37, and combinations thereof.
 5. A single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions,wherein the heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and thathas a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35,SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof.
 6. The fullyhuman single chain antibody of claim 5, wherein the single chain fullyhuman antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ IDNO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO.8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO.13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO.23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO.28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO.35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, andcombinations thereof.
 7. The fully human single chain antibody of claim5, wherein the fully human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain fully human antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO.34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO.31/SEQ ID NO. 37, and combinations thereof.
 8. A method for treating abroad spectrum of mammalian cancers or treating inflammatory diseases orautoimmune diseases, comprising administering an effective amount of ananti-CXCR3 polypeptide, wherein the anti-CXCR3 polypeptide is selectedfrom the group consisting of a fully human antibody of an IgG class thatbinds to a CXCR3 epitope with a binding affinity of at least 10⁻⁶M, afully human antibody Fab fragment, having a variable domain region froma heavy chain and a variable domain region from a light chain, a singlechain human antibody, having a variable domain region from a heavy chainand a variable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof; wherein the fully human antibody has a heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof; wherein the fully human antibody Fabfragment has the heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequences selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ IDNO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34,SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof;and wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.
 9. The method for treating a broadspectrum of mammalian cancers of claim 8, wherein the fully humanantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ IDNO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO.24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO.33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO.31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, and combinations thereof.10. The method for treating a broad spectrum of mammalian cancers ofclaim 8, wherein the fully human antibody Fab fragment has both a heavychain variable domain region and a light chain variable domain regionwherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO.32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO.37, and combinations thereof.
 11. The method for treating a broadspectrum of mammalian cancers of claim 8, wherein the fully human singlechain antibody has both a heavy chain variable domain region and a lightchain variable domain region, wherein the single chain fully humanantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ IDNO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ IDNO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ IDNO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ IDNO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ IDNO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, and combinationsthereof.
 12. The method for treating a broad spectrum of mammaliancancers of claim 8, wherein the broad spectrum of mammalian cancers tobe treated is a CXCR3-positive cancer.
 13. The method for treating abroad spectrum of mammalian cancers of claim 8, wherein the broad themammalian cancer is selected from the group consisting of is selectedfrom the group consisting of carcinomas, leukemias, lymphomas, prostatecancer, non-small cell lung cancer, breast cancer, endometrial cancer,ovarian cancer, gastric cancers, head and neck cancers, melanoma,osteosarcoma, intestinal and colon cancer, and various metastaticcancers.
 14. A method for treating an inflammatory disorder, comprisingadministering an effective amount of an anti-CXCR3 polypeptide, whereinthe anti-CXCR3 polypeptide is selected from the group consisting of afully human antibody of an IgG class that binds to a CXCR3 epitope witha binding affinity of at least 10⁻⁶M, a fully human antibody Fabfragment, having a variable domain region from a heavy chain and avariable domain region from a light chain, a single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof; wherein the fully human antibody has a heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21,SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.31, SEQ ID NO. 33, and combinations thereof, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof; wherein the fully human antibody Fabfragment has the heavy chain variable domain sequence that is at least95% identical to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ IDNO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequences selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ IDNO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34,SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof;and wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ IDNO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 33, and combinations thereof, and that has the light chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 37, and combinations thereof.
 15. The method for treating aninflammatory disorder of claim 14, wherein the fully human antibody hasboth a heavy chain and a light chain wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ IDNO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO.20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO.30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO.31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO.36, SEQ ID NO. 31/SEQ ID NO. 37, and combinations thereof.
 16. Themethod for treating an inflammatory disorders of claim 14, wherein thefully human antibody Fab fragment has both a heavy chain variable domainregion and a light chain variable domain region wherein the antibody hasa heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO.24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO.29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO.33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO.31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37, and combinations thereof.17. The method for treating an inflammatory disorder of claim 14,wherein, the fully human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain fully human antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO.21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO.26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO.34, SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO.31/SEQ ID NO. 37, and combinations thereof.
 18. The method for treatingan inflammatory disorder of claim 14, wherein, the inflammatory disorderto be treated is selected from the group consisting of rheumatoidarthritis, multiple sclerosis, Crohn's disease, Graves' disease,Sjögren's syndrome, inflammatory bowel disease, chronic obstructivepulmonary disease, psoriasis, type 1 diabetes, transplant rejectionchronic hepatitis C, malaria, and atherosclerosis.